1. Field of the Invention
The present invention relates to (1) a transglutaminase (hereinafter referred to as TG) isolated from a Bacilli such as those of Bacillus subtilis, (2) a fraction having transglutaminase activity, and (3) a method for producing a protein, a non-proteinaceous amino acid polymer, a peptide or derivatives thereof having a crosslinked structure, by crosslinking the glutamine and lysine residues in the same with the TG or the fraction having TG activity to thereby form intermolecular or intramolecular, crosslinked .epsilon.-(.gamma.-Glu)-Lys bonds.
The present invention also relates to (4) a DNA coding for a TG derived from a Bacilli such as Bacillus subtilis, (5) a vector comprising said DNA coding for said TG, (6) a cell transformed with the vector, and (7) a method for producing a Bacillus-derived transglutaminase by incubating the transformant.
The crosslinked polymers produced using the Bacillus-derived TG of the present invention can be used in foods such as tofu (soybean curd), pudding, yogurt, cheese, ground fish meat, boiled fish paste, sausage and other fish and meat products and also in cosmetics, etc.
2. Discussion of the Background
TG is an enzyme which catalyzes the transacylation of .gamma.-carboxyamide groups in the glutamine residues in a peptide chain with the either .epsilon.-amino group in a lysine residue in the peptide chain or water. When a .epsilon.-amino group is the acyl acceptor, crosslinked .epsilon.-(.gamma.-Glu)-Lys bonds (hereinafter referred to as "GL bonds") are formed in or between the peptide molecules. Where water is the acyl receptor in the transacylation, the glutamine residue in the peptide chain is subjected to de-amidation by which the glutamine residue is converted into a glutamic acid residue.
It is known that TG exists in many animal tissues. For example, TG existing in the liver of guinea pigs has been studied (see Connellan et al., J. Biol. Chem., Vol. 246, pp. 1093-1098, 1971). Microorganism-derived TGs are less well known, only TG derived from Actinomycetes (ray fungi), Bacillus subtilis (see Ramanujam et al., FASEB J. Vol. 4, A2321) and Myxomycetes (slime molds) (see Klein et al., J. Bacteriol., Vol. 174, pp. 2599-2605) have been reported. At present, TG produced by ray fungi has been put to practical and industrial use (see Japanese Patent Publication No. 6-65280, Japanese Patent Laid-Open No. 1-27471).
Unfortunately, TG derived from animals such as guinea pigs is impractical for use in industry because it is difficult to obtain a large amount of such animal-derived TG at low costs. In addition, the animal-derived TG requires calcium ions, thus limiting its use.
Ray fungus-derived TG also has some drawbacks. Since ray fungi grow more slowly than ordinary bacteria, they need a long period of time for incubation, resulting in the increase in the costs in producing TG.
Ramanujam et al. of New Mexico State University have reported the existence of Bacillus subtilis-derived TG. The TG as reported by them have the following properties:
1) The pH suitable for it is 9.5 or higher. PA1 2) Since its activity is greatly inhibited by a chelating agent (EGTA), it is considered that the TG has the property of requiring metal ions. PA1 3) It is inhibited by Ca.sup.2+ of 5 mM or more. PA1 4) It is inhibited by dithiothreitol (DTT). PA1 5) It is produced by both vegetative cells and sporulating cells. PA1 a) it is active between about pH 7 and about 9, PA1 b) it is active between about 40.degree. C. and about 65.degree. C., PA1 c) it is stable at about 60.degree. C. or lower, PA1 d) it is independent of Ca.sup.2+ and has an activity of 50% or more in the presence of 5 mM of Ca.sup.2+, PA1 e) it is inhibited by NEM, cystamine and (NH.sub.4).sub.2 SO.sub.4, PA1 f) it is not inhibited by EDTA, DTT and 2-ME, PA1 g) it has a molecular weight of (i) from about 18,000 to about 22,000 as measured by gel permeation and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, and PA1 h) it catalyzes the transacylation of the .gamma.-carboxyamide group in glutamine residue(s) in a peptide chain.
It is considered that TG reported by Ramanujam et al. is limited due to the above-mentioned properties, especially because its operating pH is high and it is influenced by metal ions.
Thus, (1) the animal-derived TG is impractical for industrial use because it requires calcium increasing production costs, (2) the ray fungus-derived TG is impractical for industrial use because the growth of ray fungi is slow increasing production costs, and (3) Ramanujam et al.'s Bacillus subtilis-derived TG is impractical for industrial use, since it cannot be used in foods since it is inhibited by 5 mM of Ca.sup.2+.